Background: Methicillin-resistant S. aureus (MRSA) infections pose an ongoing threat to human health. Though deadly MRSA infections were once most commonly associated with hospital visits, community acquired MRSA (CA-MRSA) infections are now very common. USA300 is a highly-virulent epidemic strain of CA-MRSA which is very resistant to treatment with -lactam antibiotics. Many cellular factors are required for -lactam resistance, and a better understanding of the interactions required for resistance will lead to new and effective therapeutic strategies. Preliminary Results: One of the methods for identifying genetic interactions is through next-generation sequencing (NGS) of transposon libraries. I have developed a platform for using a new high-efficiency method for making transposon libraries in S. aureus to create and sequence transposon libraries with the ability to upregulate as well as inactivate any gene in the genome. I have made and sequenced libraries in both MRSA and methicillin-sensitive (MSSA) strains of S. aureus, and I have developed the computational workflow for analyzing the results. Aim 1 is devoted to better understanding and listing all genes that when inactivated or upregulated significantly increase or decrease resistance to -lactam antibiotics. I will create a transposon library in USA300 using the platfor I developed and treat it with oxacillin and ceftobiprole. USA300 is resistant to oxacillin, so treatment with oxacillin will reveal factors that when inactivated decrease USA300 resistance. This will reveal targets for which -lactam potentiators can be developed. On the other hand, ceftobiprole can kill USA300, so treatment with ceftobiprole will uncover genes that when inactivated or upregulated result in increased resistance to the antibiotic. These results may reveal how clinical resistance will develop. A selection of these hits will be validated. Aim 2 wil focus more on better understanding peptidoglycan biosynthesis in a USA300 because as a strain of CA-MRSA, and a better understanding of peptidoglycan biosynthesis could lead to new ideas for treating these dangerous infections. I will treat the library with two -lactam antibiotcs that have a higher affinity for one penicillin-binding protein (PBP) over all the others: mecillina (PBP3) and cefoxitin (PBP4), and identify genes that when inactivated or upregulated result in a significant increase or decrease in fitness in the presence of the compound. We believe that these two PBPs are involved in two different modes of peptidoglycan biosynthesis, and by inactivating one; we may identify factors that are essential for the proper action of the other as well as identify the function of each mode of peptidoglycan biosynthesis. A selection of these genes will be prioritized for validation, and the nature of the interaction will be probed using a variety of biochemical and cell biological techniques. This approach will lead to a deeper understanding of the ways in which CA-MRSA reacts to antibiotic treatment, will shed light on the interactions required for -lactam resistance, and lead to novel therapeutic strategies.